Skin sensitization and subsequent contact allergic reaction are common in both the occupational and general population. Identification of potential allergens in consumer products has been recognized as an important step in both diagnosis and prevention of allergic contact dermatitis and other immune mediated hypersensitivity responses, including asthma. Both standardized human and animal allergen identification test have been employed and are commonly accepted. Ethics prescribed for the use of animals in consumer product safety testing dictates that appropriate alternative methods be considered and substituted for animals whenever possible. Recent advances in understanding the immune-pathological mechanisms of allergic sensitization and allergic responses have spurred research into non-animal alternative methods for identification of chemical allergens. It has been noted that most chemical allergens (or their metabolites) have electrophilic centers that can react covalently with nucleophilic protein moieties. Methods have been reported that utilize the binding of electrophilic allergens to peptide probes which are presently under review by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). As part of this research program, NIOSH has developed a rapid kinetic absorbance assay utilizing the soft nucleophile, nitrobenzene thiol (NBT), as an absorbance probe and have further characterized a hard nucleophilic probe (pyridoxylamine, PDA) that reacts with non-thiol reactive hard electrophilic allergens such as cyclic anhydrides and diones. When combined, (NBT/PDA) the assay can detect most electrophilic allergens. Covalent binding to the PDA amine produces a shift in absorbance and fluorescence that can be monitored in real time. Structural activity relationship studies using benzoquinone and substituted benzoquinones with constituents that donate or withdraw electrons to decrease or enhance electrophilicity, respectively, have been conducted to further validate the use of the NBT/PDA assay to provide potential dermal allergenic potency information. From the kinetic NBT/PDA data, end point assays have been developed that allow for identification of electrophilic allergens. The NBT/PDA assay was nominated to ICCVAM and unanimously approved by the Scientific Advisory Committee on Alternative Toxicological Methods (SACATM) at the September 5, 2012 meeting to be given a high priority for intralaboratory validation. In addition, previous research conducted under this Interagency agreement resulted in production of monoclonal antibodies (mAbs) that specifically recognize toluene diisocyanate (TDI)-haptenated proteins and methylene diphenyl diisocyanate (MDI)-haptenated proteins. These diisocyanates are the primary intermediate required for production of polyurethane lacquers, foams, binders and glues. Diisocyanates can now be found in several commercial products available to the general consumer. They are the most commonly reported cause of occupational asthma, and also are known to cause allergic contact dermatitis (ACD). Dermal exposure is thought to play a major role in sensitization to diisocyanates for both ACD and asthma, however, tools to study dermal exposure have been previously lacking. Presently these antibodies are being employed to develop biomarker assays and to evaluate the disposition of TDI following inhalation and dermal exposure. Proteomic studies to better understand diisocyanate hapten chemistry are also ongoing.